“Non-commercial” TB tests Brief overview from MSF perspective

Francis Varaine, MSFRetooling Task Force

Geneva, 15-16/01/08

Content

Microscopy• Bleach concentration• Auramine staining• FDA staining• 2 slides• Positivity threshold

Culture and DST• MODS• Thin-layer agar• Nitrate reductase• Redox indicators• Kudoh method• Slide culture• …..

Microscopy

Bleach concentration method

• Centrifugation or sedimentation increases the sensitivity of the sputum smear microscopy.

• Angeby et al., IJTLD 2004 (literature review)– Recommend the evaluation and introduction of the bleach method in

most settings where mycobacterial culture is not performed routinely.

• Steingart et al., Lancet Infect Dis. 2006 (a systematic review)– Centrifugation with any of several chemical methods (including bleach)

is more sensitive, that overnight sedimentation preceded

– There were insufficient data to determine the value of sputum processing methods in patients with HIV infection.

• Bonnet et al (MSF), CID 2008. – Bleach sedimentation: An opportunity to optimize tuberculosis smear

microscopy in high HIV prevalence settings.

Adopted, introduced and implemented

Auramine staining

• Steingart et al., Lancet Infect Dis. 2006 : Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review. – Fluorescence microscopy is more sensitive than conventional microscopy,

and has similar specificity. – There is sufficient evidence to determine the value of fluorescence

microscopy in HIV-infected individuals

• LED • Study in Kenya : bleach + auramine

Adopted, introduced and implemented

FDA staining

• FDA (fluorescein) staining can be distinguish between live and dead AFB

• For diagnosis of true treatment failure.

• A. Hamid Salim, et al., Int J Tuber Lung Dis 2006. Early and rapid microcopy-based diagnosis of true treatment failure and MDR-TB.

• In Bangladesh:

Compared FDA staining and culture on smear positive sputum.

-Sensitivity 99%

-Specificity 82%  

-Accuracy 93%                                                                       

Operational research

New definitions

• 2 slides

• Positivity threshold : – 1 positive / 2 slides– 1 AFB / 100 fields

Bonnet, et al : Reducing the number of sputum samples examined

and thresholds for positivity: an opportunity to optimise smear

microscopy. IJTLD (2007)

Adopted, introduced and implemented

Rapid culture and DST

• Microcolonies

• Colorimetric methods

MODS Microscopic observation drug susceptibility

Based on characteristic cord formation in early liquid cultures

• Caviedes et al. JCM 2000; Moore et al. JCM 2004, NEJM 2006

• MTB detection rapid (median 7 days, 13 days BACTEC, 26 days LJ)

• Sensitive (98% vs 84%)

• RIF: sensitivity 100%; specificity 100%

• INH: sensitivity 84,6%; specificity 99.6%

Caviedes et al. J. Clin Microbiol 2000

Not adopted

MODS procedure manual

http://www.ucph.edu.pe

TLA Thin Layer Agar

Based on detection of microcolonies on 7H10 agar

Characteristic “cording”

• Robledo et al., IJTLD 2006 (multicenter study)• Da Silva et. al., Braz. J. of Microbiology 2007

• Direct method for smear positive and negative specimens

• Median time to detection 10 days, for RIF and INH resistance

• Good sensitivity and specificity for direct DST (Robledo et al., submitted)

Picture: J. Robledo

Adopted for field testing

TLA multicentric study

Evaluation of a rapid culture method for tuberculosis diagnosis: a Latin American multi-center study.J. A. Robledo,G. I. Mejía, N. Morcillo,L. Chacón M. Camacho,J. Luna, J. Zurita, A. Bodon, M. Velasco, J. C. Palomino, A. Martin, F. Portaels. Int. J. Tuberc. Lung. Dis.10(6):613–619, 2006

Total of 1118 specimens studied in six sites

TLA for diagnosis

7H11 7H11 + PNB

Identification M. tuberculosis

Decontamination sputum sample NALC/NaOH

Reading with standard microscope

7H11 agar growth control

7H11 agar +PNB 500

7H11 agar + INH 0.2

7H11 agar + RIF 1

Decontamination sputum sample

Identification M. tuberculosis

Detection resistance INH Detection resistance RIF

Direct DST TLA INH/RIF

Reading with standard microscope

37°C 5%C02

+ ofloxacine under validation

Implementation of TLA in Homa Bay, Kenya (MSF)

TB culture lab.

Reading under normal microscope (objective 10x), twice a week

TLA procedure manual

http://www.itg.be

Rapid culture and DST

• Microcolonies

• Colorimetric methods

NRA (indirect)• Ängeby et al., JCM 2002, • Martin et al., JMM 2005 (multicentric study first-line drugs), • Martin et al., JCM, 2005 (ofloxacin)• INH, RIF, EMB, SM, OFLO• High accuracy (95-100%) for INH, RIF and OFLO• Median TAT: 10 days

NRA (direct) on sputum smear + samples• Musa et al. J Clin Microbiol 2005• Solis et al. Int J Tuberc Lung Dis 2005• Affolabi et al. J Clin Microbiol 2007• Median TAT: 14-28 days

Nitrate Reductase Assay (Griess method)

Considered for field testing

Study No. samples

Reference standard

INH Se/Spe

RIF Se/Spe

TAT (days)

Musa et al. 121 Direct PM 93/100 100/100 14 Solís et al. 192 Indirect PM 99/100 93/100 28*

Affolabi et al. 177 Indirect PM - 87.5/100 18

Nitrate Reductase Assay (Griess method)

Nitrate Nitrite

• Affolabi et al, JCM 2007• Performed on solid media• Rapid method for DST• Easy to implement in resource-limited countries• No need for training if implemented in lab where DST is already

performed on LJ.• No need of special equipment• Sensitivity 88%, specificity 100% • TAT: 18 days (direct)

Implementation of the NRA direct in Cotonou

Picture: Dr Affolabi, Benin

Colorimetric redox indicators

• Based on reduction of indicators by mycobacterial metabolism– Alamar Blue (Yaiko et. al JCM 1995)

– Resazurin (Palomino, AAC 2002)

• Martin et al. 2007 meta-analysis– 18 studies for RIF– 16 studies for INH

• Evidence of high sensitivity and specificity

• Other studies: EMB, SM, KAN, ETH, CAP, OFLO

Picture: A. Martin

MIC

Implementation of the resazurin in CotonouAffolabi et al., 2007 (submitted)

• Studied 151 strains of TB• Performed REMA and compared results with the gold standard PM on LJ for

RIF and INH• TAT REMA : 3 weeks + 8 days• Conventional method : 3 weeks + 4-6 weeks.

• Results – for RIF: 94% sensitivity and 100% specificity– for INH: 100 % sensitivity and 100% specificity

NRA procedure manual

http://www.itg.be

Colorimetric assay procedure manual

http://www.itg.be

Simplified culture

4% NaOH

Kudoh culture – Swab method

• Kudoh and Kudoh. Bull. World Health Org. 1974;51:71-82 : a simplified swab method for culturing mycobacteria that takes only 3-4 minutes per sample, and needs neither technical skill, centrifugation, nor a biosafety cabinet.

• Sputum is smeared onto a swab stick that is immersed into a test tube with sterile 4% sodium hydroxide.

• Very simple method to culture Mycobacteria in rural areas

• In 2006: evaluation of this method in Venezuela (Jacobus DeWaard, unpublish data)

• Kudoh swab method is as sensitive as the Petroff decontamination procedure in the diagnosis of pulmonary TB.

.

Medium Ogawa pH 6,4

• Evaluation of the Kudoh method in two hospital laboratories in Caracas, Venezuela and in the Warao Amerindian population (Orinoco River delta).

• All samples were processed for acid-fast staining and cultured using both the Petroff and Kudoh methods.

Implementation of Kudoh culture in Venezuela,Warao Amerindian population

Jacobus De Waard et al. (not yet published)

Picture: Jacobus De Waard, Venezuela

No. of sputum Samples

Acid-fast staining smear +

Culture Kudoh

Culture Petroff

Positive Total

Hospital setting

315 56 71 68 77

Field conditions

651 38 46 N.D 46

Results: Kudoh culture Jacobus De Waard et al. (unpublished)

• Kudoh swab method is as sensitive as the Petroff decontamination procedure in the diagnosis of pulmonary TB.

• It represents an alternative method for culturing mycobacteria that could reduce labor in laboratories with a high volume of sputum samples, especially appropriate for rural settings without the proper infrastructure for traditional culturing.

• The method needs no laboratory equipment, and the minimal sample manipulation reduces biosecurity risks, so that it can be easily taught to technicians without experience in tuberculosis microbiology.

Considered for operational research

Slide DST

Slide DST

• Culture perform directly on slide smear-positive samples

• Counting acid-fast micro-colonies under microscope

• A. Hamid Salim et al., Int J Tuber Lung Dis 2006. Early and rapid microcopy-based diagnosis of true treatment failure and MDR-TB.

• Sensitivity and specificity RIF:96%

• Sensitivity and specificity INH: 99% and 64%

Picture: A. van Deun

Considered for operational research

Early and rapid microcopy-based diagnosis of true treatment failure and MDR-TB, 2006, Int J Tuber Lung Dis 10 (11):1248-1254. A. Hamid Salim, et al

Summary

Microscopy• Bleach concentration• Auramine staining• FDA staining• 2 slides• Positivity threshold

Culture and DST• MODS• Thin-layer agar• Nitrate reductase• Redox indicators• Kudoh method• Slide culture

ImplementedImplementedOperational researchImplementedImplemented

Not adoptedAdopted for field testConsidered for field testNot adoptedConsidered for operational researchConsidered for operational research

Conclusion

• Some tools exist– Some ready for implementation– Some require more field testing– Some require more evidence

• Why so slow ? • Why so neglected ?